Biology Reference
In-Depth Information
vessel and let the reaction proceed for 1 h. Repeat the FITC
coupling.
9. Repeat Steps from Subheading 2.2 , step 4 to Subheading 3.2 ,
step 10 .
10. HPLC purify the conjugate.
11. Check the identity of the molecule by mass spectroscopy and
its purity by HPLC.
1. Culture human leukemia K562 cells in humidifi ed atmosphere
of 5 % CO 2 /air in RPMI 1640 medium supplemented with
10 % fetal bovine serum, 50 U/mL penicillin, and 50
3.4 Human Cell Lines
and Culture Conditions
μ
g/mL
streptomycin [ 15 ].
2. Prepare a stock solution of Mithramycin (MTH) 100
M;
store the solution at −20 °C in the dark and dilute it immedi-
ately before the use [ 15 , 16 ].
3. Treat the K562 cells with 15 nM of Mithramycin (MTH) at
the beginning of the cultures (seed 30,000 cells /mL).
μ
1. Dissolve the lyophilized PNA in ultrapure water at a 500
μ
M
3.5 Uptake
Evaluation by FACS
concentration.
2. Incubate the cells 3 × 10 4 cell/mL (K562 previously treated
with mithramycin) with and without 2
μ
M of the PNA-peptide-
FITC conjugate for 48 h.
3. Harvest and wash the cells.
4. Analyze 1 × 10 5 cells by the CellQuestTM version 3.3 software
(Becton Dickinson), using the FL1 channel to detect
fl uorescence.
5. Repeat the experiment at least 3 times for statistical analysis.
6. Express the results as median fold, i.e., the ratio between the
median fl uorescence intensity values obtained in the presence
and absence of treatment, respectively. Data can be presented
with an histogram, showing the number of cells versus the
expressed fl uorescence intensity.
An example of FACS data is reported in Fig. 2 for K562 cells
treated with FITC-PNA-peptide conjugates.
1. Isolate cells by centrifugation at 1,500 rpm (1,600 × g ) for
10 min at 4 °C and wash in PBS 1×.
2. Lyse the cells with Tri-Reagent TM , according to manufacturer's
instructions.
3. Precipitate the isolated RNA in 75 % cold ethanol and store it
at −80 °C.
4. Dry and dissolve RNA in ultrapure nuclease-free water before
use.
3.6 RNA Extraction
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